|Rodriguez, Lorena -|
|Martinez, Beatriz -|
|Zhou, Yuan -|
|Rodriguez, Ana -|
|Garcia, Pilar -|
Submitted to: BioMed Central Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 20, 2011
Publication Date: March 20, 2011
Citation: Rodriguez, L., Martinez, B., Zhou, Y., Rodriguez, A., Donovan, D.M., Garcia, P. 2011. Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. BioMed Central Biology. 11:138-145. Interpretive Summary: Problem-- Staphylococcus aureus is a notorious mastitis pathogen for which conventional antibiotics are less than 50% effective. There is a need for new antimicrobials for Staphylococcus aureus. Phage lysins are strong candidate antimicrobials due to their near-species specificity and thus reduced risk of resistance development, compared to broad range antibiotics. Accomplishment -- A novel phage lytic protein HydH5 was isolated and shown to have antimicrobial properties towards mastitis causing S. aureus. Contribution of Accomplishment to Solving the Problem—Identification and antimicrobial characterization of novel staphylolytic enzymes is the first step toward commercialization. This enzyme is unusually thermostable, suggesting that it might have other applications in food safety e.g. active following pasteurization procedures.
Technical Abstract: Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88) contains a virion-associated muralytic enzyme (HydH5) encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encoded by staphylococcal phages. The protein consists of 634 amino acid residues. Two putative lytic domains were identified: an N-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain (135 amino acid residues) , and a C-terminal LYZ2 (lysozyme subfamily 2) domain (147 amino acid residues) . Predicted three-dimensional structure of HydH5 confirmed these domains and provided the basis for deletion analysis. The complete HydH5 protein and truncated proteins containing only each catalytic domain were overproduced in E. coli and purified from inclusion bodies by subsequent refolding. Truncated and full-length HydH5 proteins were all able to bind and lyse S. aureus Sa9 cells as shown by binding assays, zymogram analyses and viability tests. HydH5 demonstrated high antibiotic activity against early exponential cells, at 45ºC and in the absence of divalent cations (Ca2+, Mg2+, Mn2+). Thermostability assays showed that HydH5 retained 72% of its activity after 5 min at 100°C. Based on these results, this protein is proposed as a potential new antimicrobial against S. aureus.